This invention relates to methods for preserving solutions and suspensions containing biologically active molecules, viruses (vaccines), cells, and small multicellular specimens. More particularly, the invention relates to methods for long-term storage of these labile biological materials at ambient temperatures in dehydrated, very viscous amorphous liquid or glass state.
The preservation and storage of solutions or suspensions of biologically active materials, viruses, cells and small multicellular specimens is important for food and microbiological industries, agriculture, medical and research purposes. Storage of dehydrated biologically active materials carries with it enormous benefits. Dehydrated reagents, materials and cells have reduced weight and require reduced space for storage, notwithstanding their increased stability.
Suggestions in the prior art for providing enhanced-stability preparations of labile biological materials in dehydrated form include freeze-drying and vacuum or air-drying. While freeze-drying methods are scalable to industrial quantities, materials dried by such methods can not be stored at ambient temperatures for long periods of time. In addition, the freezing step of freeze-drying is very damaging to many sensitive biological materials. Alternatively, vacuum and air-drying methods do not yield preparations of biological materials which are scalable to industrial quantities and stable for extended periods of time at ambient temperatures, because destructive chemical reactions may continue to proceed in such dried preparations.
Some of the problems associated with preservation by freezing and drying have been addressed by addition of protectant molecules, especially carbohydrates, which have been found to stabilize biological materials against the stresses of freezing and drying. However, despite the presence of protectants, the long-term stability may still require low temperature storage, in order to inhibit diffusion-dependent destructive chemical reactions. Thus, further innovations have been sought to provide long-term storage of labile biological materials at ambient temperatures.
Storage of dried materials at ambient temperatures would be cost effective when compared to low temperature storage options. Furthermore, ambient temperature storage of biological materials such as vaccines and hormones would be extremely valuable in bringing modern medical treatments to third world countries where refrigeration is often not available. As the many benefits of shelf preservation of biological specimens have come to be appreciated, researchers have endeavored to harness vitrification as a means of protecting biological materials against degradative processes during long-term storage. Consequently, this technology of achieving the xe2x80x9cglassxe2x80x9d state, has been anticipated to emerge as a premier preservation technique for the future.
A glass is an amorphous solid state which may be obtained by substantial undercooling of a material that was initially in the liquid state. Diffusion in vitrified materials, or glasses, occurs at extremely low rates (e.g. microns/year). Consequently, chemical or biological changes requiring the interaction of more than one moiety are practically completely inhibited. Glasses normally appear as homogeneous, transparent, brittle solids, which can be ground or milled into a powder. Above a temperature known as the glass transition temperature (Tg), the viscosity drops rapidly and the glass becomes deformable and the material turns into a fluid at even higher temperatures. The optimal benefits of vitrification for long-term storage may be secured only under conditions where Tg is greater than the storage temperature. The Tg is directly dependent on the amount of water present, and may therefore be modified by controlling the level of hydration; the less water, the higher the Tg.
Unfortunately, the advantages of vitrification technology as a means of conferring long-term stability to labile biological materials at ambient temperatures have not been fully utilized. Current methods of ambient temperature preservation by drying are designed for laboratory processing of very small quantities of materials. Consequently, such methods are not compatible with large scale commercial operations. Other technical problems related to monitoring of the glass transition temperature have also posed obstacles to the commercial development. Thus, while drying and vitrification technology are potentially attractive as scalable methods for long-term storage of biological materials, problems remain to be overcome before the advantages of storage in the glass state can be commercially exploited.
A method is disclosed for preserving industrial quantities of solutions and suspensions containing sensitive biological materials comprising drying the samples by boiling under vacuum in a temperature range of xe2x88x9215xc2x0 C. to 70xc2x0 C. A mechanically-stable foam, consisting of thin amorphous films of concentrated solutes is formed. Such foams will not collapse for a least one hour at xe2x88x9220xc2x0 C. when stored under vacuum. To increase the stability, the foams can be further dried for at least 12 more hours under vacuum at temperatures ranging from 0xc2x0 to 100xc2x0 C., wherein the drying temperature is greater than the desired storage temperature, selected from within the range of 0xc2x0 to 70xc2x0 C.
To provide long-term shelf preservation of biological solutions and suspensions in the glass state, the mechanically-stable foams may be subjected to secondary drying under vacuum in the range of 0xc2x0 to 100xc2x0 C. for a period of time sufficient to increase the glass transition temperature to a point above the selected storage temperature within the range of 0xc2x0 to 70xc2x0 C. Finally, a composition is disclosed for protecting cells and viruses during the recited drying and vitrification processes, comprising a non-reducing monosaccharide, a dissacharide (like sucrose) and a biological polymer.